Title Defining the Niche-dependent Role of RNA Editing in Aged and MDS Hematopoietic Stem and Progenitor Cell Dysfunction Project Summary During aging, impaired hematopoietic stem and progenitor cell (HSPC) maintenance induced by clonal DNA mutations as well as niche-driven RNA processing deregulation can set the stage for myelodysplastic syndrome (MDS) initiation. Recently, increased adenosine deaminase associated with RNA1 (ADAR1)-mediated A-to-I editing was shown by our group and other research teams to contribute to therapeutic resistance in a broad array of malignancies. Also, we discovered that lentivirally enforced ADAR1 expression in HSPC enhanced myeloid differentiation commensurate with upregulation of PU.1 and reduced dormancy. Whole transcriptome RNA sequencing (RNA-seq) analysis demonstrated that inflammatory cytokine signaling pathways and RNA editing increased during normal aged HSPC evolution to MDS. Thus, we hypothesized that niche dependent activation of RNA editing by ADAR1 provides a competitive advantage for MDS over normal HSPCs. The majority of ADAR1 mediated adenosine-to-inosine (A-to-I) RNA editing events in humans occur within double- stranded RNA (dsRNA) loops created by primate-specific Alu sequences, which comprise 10 percent of the human genome, thereby underscoring that important ADAR1 functional differences exist between human HSPCs compared with their murine counterparts. However, the limited research effort aimed at deciphering the role of ADAR1-mediated RNA editing in HSPC maintenance has been performed primarily in mouse models rather than highly purified human HSPCs. Because ADAR1 is activated by inflammatory cytokines that accelerate aging and MDS initiation, our main goal is to define the niche- dependent role of RNA editing on human HSPC cell fate and cell cycle regulation during aging and MDS initiation. We will first determine the RNA editing profile by whole transcriptome and single cell RNA-seq, RESSqPCR and lentiviral RNA editing reporters. The functional role of ADAR1 in HSPC aging and MDS initiation will be examined in stromal co-cultures with or without addition of inflammatory cytokines, FUCCI2BL cell cycle reporters, and humanized aged HSC and MDS immunocompromised mouse models. We will also examine the effect of RNA editing on APOBEC3 family of DNA deaminase function during MDS initiation. The proposed study is uniquely responsive to PAS-13-033: Stimulating Hematology Investigation: New Endeavors (SHINE) because it will identify the role of ADAR1-mediated regulatory mRNA and miRNA editing in HSPC myeloid lineage commitment and cell cycle deregulation during age-dependent MDS initiation in the inflammatory bone marrow niche. The ultimate goal of this study is to determine the biological, diagnostic and prognostic significance of ADAR1-mediated RNA editing in HSPC aging compared with MDS initiation.